This proposal aims at elucidating the epigenetic control of embryonic stem cell (ESC) commitment to the skeletal muscle lineage and further maturation into differentiated myotubes in response to the extra-cellular signal-activated p38 pathway. We will elucidate the mechanism by which the SWl/SNF chromatin remodeling component BAF60c (encoded by SMARCD3) enables MyoD to convert ESCs into the myogenic lineage and will characterize the molecular basis by which the p38 signaling coordinates gene activation and repression in ESC-derived muscle progenitors through the genome re-distribution of two distinct chromatin-modifying complexes - the SWI/SNF and the Polycomb group (PcG) complexes. We will also perform high-throughput screening (HTS) to identify compounds that sensitize ESCs to adopt the skeletal muscle lineage, via induction of endogenous BAF60c expression. The following are specific aims. Aim 1. To elucidate the mechanism by which BAF60c enables MyoD to convert ESC into muscle progenitors (ESC BAF60c/MyoD). 1a- Perform gene expression profiling and genome-wide chromatin analysis in hESCs committed to the myogenic lineage by the ectopic expression of MyoD and BAF60c. 1b. Characterize the function of BAF60c in the myogenic commitment of hESCs. Aim 2. To characterize the response of ESC BAF60c/MyoD to the p38 signaling. 2a- Analyze the p38-mediated recruitment of SWI/SNF complex to the chromatin of muscle differentiation genes in ESC BAF60c/MyoD. 2b Analyze the p38-dependent repression of Pax7 in ESC BAF60c/MyoD. Aim 3. To identify compounds that implement ESC conversion to the myogenic lineage, via BAF60c induction. 3a- Analyze the epigenetic profile of the BAF60c locus and characterize the BAF60c promoter during hESC transition to muscle progenitor cells. 3b- Use BAF60c promoter-GFP reporter and high- throughput screening (HTS) to identify compounds that induce BAF60c in ESCs. We anticipate that the knowledge gained from this research will contribute to identify strategies that implement stem cell-based regeneration of diseased muscles. Overall, our proposed research will reveal targets for novel interventions directed to the interface between intracellular signaling pathways and downstream epigenetic modifications, for a pharmacological control of muscle stem cells. PUBLIC HEALTH RELEVANCE: This proposal aims at elucidating the epigenetic basis that control human embryonic stem cell (hESC) commitment to the skeletal myogenic lineage, their further maturation into differentiated muscles, and their response to extra-cellular signals. The ultimate goal of this proposal is to identify novel targets for strategies that manipulate stem cells to regenerate diseased muscles. We predict that the results gathered from this proposal will contribute to fill the gap between our current knowledge of stem cell biology and the mechanism by which stem cells reprogram their genome toward specific cellular lineages in response to extrinsic signals. This information is critical for the pharmacological manipulation of hESC to generate tissue progenitors and devise strategies in regenerative medicine, such as stem cell-based regeneration of diseased muscles. An additional benefit that will derive from this proposal is the potential discovery of the epigenetic basis underlying the different responsiveness to external cues in hESC-derived muscle progenitors vs adult muscle stem cells (satellite cells). This comparison will help to optimize regenerative strategies from exogenous (hESC) or endogenous (satellite cells) sources of muscle progenitors.